Decreased binding capacity (Bmax) of muscarinic acetylcholine receptors in fibroblasts from boys with attention-deficit/hyperactivity disorder (ADHD)

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Johansson, J., Landgren, M., Fernell, E., Lewander, T., Venizelos, N. (2013)

Decreased binding capacity (Bmax) of muscarinic acetylcholine receptors in fibroblasts from

boys with attention-deficit/hyperactivity disorder (ADHD).

ADHD Attention Deficit and Hyperactivity Disorders, 5(3): 267-271

http://dx.doi.org/10.1007/s12402-013-0103-0

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S H O R T C O M M U N I C A T I O N

Decreased binding capacity (B

max

) of muscarinic acetylcholine

receptors in fibroblasts from boys with attention-deficit/

hyperactivity disorder (ADHD)

Jessica Johansson•Magnus Landgren• Elisabeth Fernell• Tommy Lewander• Nikolaos Venizelos

Received: 12 November 2012 / Accepted: 22 January 2013 / Published online: 7 February 2013 Ó The Author(s) 2013. This article is published with open access at Springerlink.com

Abstract Monoaminergic dysregulation is implicated in attention-deficit/hyperactivity disorder (ADHD), and methylphenidate and amphetamines are the most fre-quently prescribed pharmacological agents for treating ADHD. However, it has recently been proposed that the core symptoms of the disorder might be due to an imbal-ance between monoaminergic and cholinergic systems. In this study, we used fibroblast cell homogenates from boys with and without ADHD as an extraneural cell model to examine the cholinergic receptor density, that is, musca-rinic acetylcholine receptors (mAChRs). We found that the binding capacity (Bmax) of [3H] Quinuclidinyl benzilate (3H-QNB) to mAChRs was decreased by almost 50 % in the children with ADHD (mean = 30.6 fmol/mg protein, SD = 25.6) in comparison with controls [mean = 63.1 fmol/mg protein, SD = 20.5, p B 0.01 (Student’s unpaired t test)]. The decreased Bmaxindicates a reduced cholinergic

receptor density, which might constitute a biomarker for ADHD. However, these preliminary findings need to be replicated in larger ADHD and comparison cohorts. Keywords ADHD Muscarinic acetylcholine receptors  Receptor binding assay  Fibroblasts  Biomarker

Introduction

Attention-deficit/hyperactivity disorder (ADHD) is the most common neurodevelopmental disorder, with a worldwide prevalence of about 5–7 % in childhood (Faraone et al.2003; Polanczyk et al.2007). The catecholaminergic systems are implicated in the pathophysiology of ADHD, and the phar-macological treatment for ADHD includes central stimu-lants that enhance the neurotransmission of dopamine and norepinephrine through multiple actions. Interactions between the dopaminergic and the serotonergic neurotrans-mitter systems suggest that serotonin, in addition to dopa-mine, is likely to be linked to the disorder (Oades2008).

Recently, it was suggested that the core symptoms of ADHD might be attributable to an imbalance between the neuromodulatory effects of monoamines and acetylcholine (Ach), implicating a monoaminergic–muscarinic receptor interaction (Vakalopoulos 2007). ACh plays an important role in regulating central nervous system (CNS) functions, including memory, attention and motivation processes—all of which have been found to be dysregulated in ADHD. Improvement of ADHD symptoms has been reported in adults and youths treated with cholinergic enhancers, such as Ach partial agonists (e.g. ABT-089) and acetylcholin-esterase (AChE) inhibitors (e.g. donepezil) (Doyle et al.

2006; Wilens et al.2006), although results are inconclusive (Cubo et al.2008). Atomoxetine, a non-stimulant drug used J. Johansson N. Venizelos (&)

Department of Clinical Medicine, School of Health and Medical Sciences, O¨ rebro University, 701 82 O¨rebro, Sweden

e-mail: nikolaos.venizelos@oru.se M. Landgren E. Fernell

Unit of Neurodevelopmental Disorders, Department

of Paediatrics, Skaraborg Hospital, 542 24 Mariestad, Sweden E. Fernell

The Research and Development Centre, Skaraborg Hospital, 541 85 Sko¨vde, Sweden

E. Fernell

The Gillberg Neuoropsychiatry Centre, Sahlgrenska Academy, Gothenburg, Sweden

T. Lewander

Department of Neuroscience, Psychiatry, Uppsala University Hospital, 750 17 Uppsala, Sweden

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for treating ADHD has been found to increase cholinergic neurotransmission in animals, indicating a procholinergic profile of this drug (Tzavara et al.2006).

With respect to ADHD neurotransmission research, there is a need for extraneural patient-derived cell models and biochemical markers that are detectable and quantifi-able in easily accessible tissues, but still reflecting neuronal conditions. One such possible cell model is primary skin fibroblasts that are being used for investigating biochemi-cal differences in various neuropsychiatric disorders, such as Parkinson’s disease, schizophrenia and Alzheimer’s disease (Auburger et al. 2012; Mahadik and Mukherjee

1996; Vestling et al. 1995). Recent studies have also demonstrated that fibroblast cells can be directly converted into functional neurons, which further can be directed towards distinct functional neurotransmitter phenotypes (Pfisterer et al.2011; Vierbuchen et al.2010).

Skin fibroblasts represent a model of primary human cells and comprise the polygenic risk factors of specific patients. This model has many advantages compared to other peripheral cell models (e.g. platelets and lympho-cytes), one being that after several passages in culture, fibroblasts are free from state-related changes and effects of medications, drugs and hormones, and thus patients do not have to be unmedicated at the time of the biopsy col-lection (Mahadik and Mukherjee1996).

Given the relevant impact of cholinergic dysregulation in ADHD, the aim of the present study was to examine the density of muscarinic acetylcholine receptors (mAChRs) in fibroblasts from children with ADHD, and in a comparison group, in an in vitro receptor binding assay.

Materials and methods

The study was approved by the Regional Ethics Committee in Gothenburg, Sweden. A written and signed consent was obtained from the parent/s and their child before per-forming the studies.

Subjects

The index group consisted of fibroblast cell lines from 11 boys with ADHD of the combined type (inattention com-bined with hyperactivity and impulsivity) according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) (APA 1994). They were between 10 and 11 years of age (mean 10.5 years) and treated with either methylphenidate or atomoxetine. They were consecutively recruited from a specialized paediatric unit for children and adolescents with ADHD at Skaraborg’s hospital in the south-western part of Sweden. With respect to aetiology, at least nine of the 11 boys had a clear hereditary origin, that

is, had first-grade relatives with ADHD and uneventful pregnancy and perinatal periods. Children with intellectual disability or meeting full criteria for autism spectrum dis-orders were excluded from the study. The comparison group comprised cell lines from nine boys, between 7 and 12 years (mean 9 years), without any diagnosis of a neu-rodevelopmental disorder. This group was recruited in conjunction with surgery at the ear, nose and throat clinic at Skaraborg’s hospital.

Cell culture

In order to prepare fibroblast cell homogenates to be used in the receptor binding assays, each cell line was seeded in T75 tissue culture flasks (Costar Europe Ltd, Costar NY, USA) containing minimal essential medium (MEM, sup-plemented with 10 % foetal bovine serum (FBS, GibcoÒ),

L-glutamine (2 mM/L), penicillin (100 mg/mL),

strepto-mycin (100 mg/mL) and Amino-MaxTM(0.06 % vol/vol). All growth media, antibiotics and FBS were obtained from Invitrogen, Life Technologies Europe BV, Sweden. The cells were incubated at a temperature of 37°C in the presence of 5 % CO2 and high humidity. When the cells reached confluence, they were harvested by trypsinization, washed twice with phosphate-buffered saline [PBS, National Veterinary Institute (SVA), Sweden] and diluted in 1.5 mL of sterile Milli Q water. Protein concentrations for each cell homogenate were determined by the Bradford method, using bovine serum albumin as a standard (approximately 1 mg/mL of cell protein per cell line was used in the receptor binding assay). The cell homogenates were stored at -20°C until used in the receptor binding assay. Cell lines between 7th and 15th passages (i.e. the number of splitting) were used in the experiments. Receptor binding assay

The receptor binding assay in fibroblast cell homogenate was done according to the modified procedure of Vestling et al. (1995). This was done by adding 25 lL of different concentrations (i.e. 0.1–3.0 lM) of [3H] Quinuclidinyl benzilate (3H-QNB, specific activity 50.5 Ci/mmol, Larodan Fine Chemicals AB, Sweden) into plastic tubes, containing 200 lL sodium phosphate buffer (pH 7.4). Half of the tubes contained unlabelled atropine (1.0 9 10-4 lM, Sigma-Aldrich, USA) to correct for non-specific binding. Fibroblast cell homogenates (25 lL; protein concentrations approximately 1 mg/mL) were added to the tubes, making a total volume of 250 lL. Tubes were incubated at 22°C for 60 min and filtered rapidly through Whatman GF/C glass fibre filters (Fisher Scientific, Sweden) presoaked for 50 min in 0.05 % poly-ethyleneimine solution. The filters were washed twice

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under vacuum with ice-cold sodium phosphate buffer. The filters were then placed in scintillation vials to which scintillation liquid was added (Optiphase Hisafe 3, Perk-inElmer Life Science, USA) and the disintegrations per minute (DPM) was counted by a scintillation counter (Winspectral 1414, PerkinElmer Life Science, USA). Calculations and statistical analysis

The specific binding of 3H-QNB was calculated by sub-tracting the values for non-specific binding in the presence of unlabelled atropine from those of total binding in the absence of atropine. All incubations were performed in duplicates and mean values were used for the calculation of binding characteristics. The binding capacity (Bmax) and the equilibrium dissociation constant (KD) of mAChR antagonist QNB were determined by nonlinear regression analysis.

Assumptions about parametric methods were met for KD of ADHD patients, but not for KDof controls or for Bmaxof ADHD patients and controls (determined by the D0Agostino and Pearson omnibus normality test). Therefore, the non-parametric Mann–Whitney U test was used to compare the Bmaxand KDvalues between patients and controls. However, three outliers were identified by the scaled Median Absolute Deviation (MADE) method and by Tukey’s method; one in

the comparison group and two in the ADHD group. If excluding these outliers, assumptions about parametric methods were met and Student’s unpaired t test was used to compare the Bmaxand KDvalues between patients and con-trols. For all statistical analyses, a significant level of 5 % was accepted and all calculations and analyses were per-formed using GraphPad Prism version 5 for Windows.

Results

Children with ADHD had a significantly lower binding capacity (Bmax) of the mAChR ligand QNB in comparison with controls (p B 0.01) when excluding the three outliers identified by the MADE method. The KD did not differ significantly between the two groups. These results suggest that the children with ADHD had a reduced density (*50 %) of mAChRs in fibroblasts, but that the affinity of 3H-QNB binding to the cholinergic receptors was not affected (Fig.1).

Discussion

The main finding in the present study was that children with ADHD had a significantly lower Bmaxof the mAChRs Fig. 1 Binding characteristics of [3H] Quinuclidinyl benzilate

(3H-QNB) binding to muscarinic acetylcholine receptors (mAChRs) in fibroblasts from children with attention-deficit/hyperactivity disor-der (ADHD). a Illustrates the individual and median (horizontal bars) Bmax and KD values of mAChRs in fibroblasts from children with ADHD (n = 11) and controls (n = 9), including outliers. *Outliers,

identified by the scaled Median Absolute Deviation (MADE) method. bIllustrates the individual and mean (horizontal bars) Bmaxand KD values of mAChRs in fibroblasts from children with ADHD (n = 9) and controls (n = 8), excluding outliers. Bmax indicates binding capacity, KD indicates the equilibrium dissociation constant and mAChRs indicates muscarinic cholinergic receptors. **p B 0.01

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ligand QNB in comparison with controls. These results suggest that the children with ADHD had a reduced density of mAChRs in fibroblasts, which potentially could be due to genetic (e.g. mutation/s in the genes coding for mAChRs) and/or post-transcriptional (e.g. mRNA stability) factors. To our knowledge, similar findings have not been reported previously. However, three outliers were identi-fied and we can only speculate about the reason for these extreme values, as they were not caused by experimental errors. The two ADHD outliers may represent different subgroups of ADHD and at least one of these boys had no evidence of hereditary origin. Since ADHD is a very het-erogeneous disorder with regard to molecular genetics and phenotypic diversity (Thome et al.2012), the finding may nevertheless be of relevance. However, further studies including different subgroups of ADHD are needed in order to draw firm conclusions.

We used fibroblast cells, derived from skin biopsies, obtained from children with ADHD and from controls, since these cells are considered to be a relevant experi-mental model for functional studies in humans (Stahl1985; Auburger et al.2012). Despite our finding of a difference in the density of mAChRs in fibroblasts of children with ADHD and controls, we can only speculate that the density of mAChRs is also decreased in the CNS. Until now, to our knowledge, no studies on mAChRs density have been performed in vivo in children with ADHD.

In a recent study, muscarinic cholinergic receptor binding (I-MR) was found to be lower (determined in lymphocytes) in children with ADHD (Coccini et al.2009). This is in accordance with our findings; however, in that study, the I-MR was only significantly decreased in girls with ADHD, but not in boys. No explanation/theory for these gender differences was given.

We are aware of the limitations of our study, such as a small sample size, a small comparison group and the index group consisting of only boys. The identification of outliers would possibly be explained by the large heterogeneity and complexity of molecular factors underlying ADHD. Therefore, the results should be considered as preliminary. Future studies have to include larger sample size consisting of both boys and girls with hereditary and non-hereditary ADHD.

We conclude that fibroblasts derived from patients with ADHD offer a useful model for exploring neurological aspects in vitro, thus allowing new hypothesis and drugs to be tested. The indicated reduced density of mAChRs in fibroblasts from children with ADHD may constitute a biomarker for ADHD; however, these preliminary findings need to be replicated.

Acknowledgments To the memory of Dr. Christer Larsson, his contributions to this field of research will always be remembered. The

authors are grateful to all children participating in the study. We also acknowledge Ivo Bate and Shahida Hussain for taking part in some of the experiments during their course work at the Neuropsychiatric Research Laboratory, O¨ rebro University. The study was supported by the grants from the Research and Development Centre Skaraborg Hospital (FoU center Dr 91021), the Swedish Research Council (K2007-62X-08318-20-3) and Fredrik and Ingrid Thurings foundation.

Conflict of interest The authors declare that they have no conflict of interest.

Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, dis-tribution, and reproduction in any medium, provided the original author(s) and the source are credited.

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